红景天苷对缺氧肾小管上皮细胞Toll受体2、IL-6表达及其凋亡的影响

The Influences of Salidroside on the Expressions of TLR-2 and IL-6 and the Apoptosis of HKC Induced by Hypoxia

目的:观察红景天苷(salidroside Sal.)对氯化钴(cobaltous chloride,Co Cl2·6H2O)诱导的缺氧人肾小管上皮细胞(human kidney tubular epithelia cell,HKC)Toll样受体2(toll-like receptor 2,TLR-2)表达、白细胞介素-6(interleukin-6,IL-6)的分泌及细胞凋亡的影响。方法:HKC细胞置于六孔板培养,随机分为正常对照组、缺氧模型组、红景天苷(salidroside Sal.)干预组(25μg/ml、50μg/ml、100μg/ml)。其中缺氧模型采用300μmol/L氯化钴处理6 h得到,红景天苷干预组在此模型基础上再用不同浓度的红景天苷处理缺氧细胞24 h。RT-PCR和Western blot检测TLR-2的表达,ELISA检测各组细胞上清中IL-6含量,流式细胞仪检测缺氧HKC凋亡情况。结果:氯化钴诱导HKC TLR-2蛋白(P<0.05)及RNA表达上调(P<0.01)、IL-6分泌增加(P<0.01)及细胞凋亡增加(P<0.01)。25μg/ml和50μg/ml的红景天苷能降低缺氧HKC细胞IL-6的水平(P<0.01),50μg/ml红景天苷抑制作用强于25μg/ml(P<0.05)。与缺氧模型组比较,尽管100μg/ml红景天苷能降低缺氧HKC细胞IL-6的绝对值水平,但差异无统计学意义(P>0.05)。25μg/ml,50μg/ml和100μg/ml红景天苷均能抑制TLR-2蛋白表达(P<0.01),且100μg/ml较25μg/ml红景天苷抑制作用显著(P<0.05),但25μg/ml与50μg/ml、50μg/ml与100μg/ml红景天苷比较,它们抑制作用的强弱差异无统计学意义(P>0.05)。三种浓度的红景天苷也能抑制缺氧HKC TLR-2的RNA表达(P<0.01),50μg/ml及100μg/ml红景天苷抑制作用强于25μg/ml红景天苷(P<0.05),但50μg/ml与100μg/ml红景天苷比较,二者抑制作用差异无统计学意义(P>0.05)。50μg/ml和100μg/ml红景天苷能抑制缺氧HKC凋亡(P<0.01),但25μg/ml红景天苷未表现出抑制凋亡的作用(P>0.05),50μg/ml与100μg/ml红景天苷比较,两者抑制凋亡的作用差异无统计学意义(P>0.05)。结论:红景天苷对缺氧HKC的TLR-2蛋白及RNA表达、IL-6的分泌及细胞凋亡均有抑制作用,但其抑制效应呈现非剂量依赖性。

Objective： To observe the effect of salidroside （ sal. ） on expressing of TLR - 2, secreting of IL - 6 and apoptosis of hypoxic human kidney tubular epithelial cell （HKC） which is induced by cobaltous chloride. Methods：HKC were put in six well plates, and randomly divided into normal group, hypoxia group, hypoxia ＋ 25 μg/ml sal. group, hypoxia ＋ 50 μg/ml sal. group and hypoxia ＋ 100 μg/ml sal. group. Cobaltous chloride was used to mimic the hypoxia condition in chronic kidney disease（CKD） in the initial stage of CKD for HKC, and its optimal concentration was established as 300μmol/L. The hypoxic model group was obtained by treating with 300 μmol/L cobaltous chloride for 6 hours, and the Sal. - intervened group was treated with different concentrations of salidroside for 24 hours on the basis of the hypoxic model. RT - PCR and Western blot were used to measure the expression of RNA and protein of TLR -2 respectively, ELISA tO detect the level of IL- 6 in the supernatant of each group, and flow cytometry （FCM） to detect apoptosis of hypoxic HKC. Results： Cobaltous chloride induced HKC to up - regulate the expressions of protein and RNA for TLR - 2 （P 〈 0. 05, P 〈 0.01, respectively）, increase the level of IL - 6 （ P 〈 0.01 ）, and promote the apoptosis （ P 〈 0.01 ）. 25μg/ml and 50 μg/ml salidroside significantly reduced the expression of IL -6 in hypoxic HKC （P 〈 0.01 ） and the inhibitory effect of 50 μg/ml salidroside was stronger than that of 25 p,g/ml salidroside （ P 〈 0.05）. In comparison to the hypoxia model group, 100 μg/ml salidroside had no significantly inhibitory effect although it ean decrease the absolute level of IL - 6 in hypoxic HKC （ P 〉 0.05）. 25μg/ml, 50 p,g/ml and 100 μg/ml salidroside all can downregulate the expression of TLR-2 protein in each cobaltouschloride -induced HKC group （P 〈 0.01 ） and there was statistical difference in this inhibitory effect when 25 μg/ml VS 100 tLg/ml salidroside （P 〈 0.05 ）, however, there was no statistical difference when 25 μg/ml vs 50 μg/ml, 50 μg/ml vs 100 μg/ml salidroside （ P 〉 0.05 ）. Above three concentrations of salidroside also decreased the expression of TLR - 2 RNA in the hypoxic HKC （ P 〈 0.01 ） and there was statistical difference in this inhibitory effect when 25 μg/ml vs 50 μg/ml, 25 μg/ml vs 100 μg/ml salidroside （P 〈 0.05 ）, but there was no statistical difference when 50 μg/ml vs 100 μg/ml salidroside （P 〉 0.05 ）. 50 μg/ml and 100 μg/ml salidroside can significantly reduce the apoptosic rate of HKC in hypoxia （ P 〈 0.01 ） , while 25 ~g/ml salidroside had no this inhibitory effect （ P 〉 0.05 ）. There was no statistical difference in depressing apoptosis for hypoxic HKC when 50 μg/ml vs 100 μg/ml sali-droside （ P 〉 0.05 ）. Conclusion： Salidroside can inhibit the expressions of both protein and RNA of TLR - 2 in hypoxic HKC, reduce the level of IL - 6, and attenuate the apoptosis for hypoxic HKC. However, these inhibitory effects of salidroside are in a dose - independent manner.